TREATMENT OF SULFONAMIDE-RESISTANT TREATMENT OF SULFONAMIDE-RESISTANT FURUNCULOSIS IN TROUT AND...
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UNITED STATES DEPARTMENT OF THE INTERIOR, Fred A. Seaton, Secretary
FISH AND WILDLIFE SERVICE, Arnie J. Suomela, Commissioner
TREATMENT OF SULFONAMIDE-RESISTANT FURUNCULOSIS
IN TROUT AND DETERMINATION OF DRUG SENSITIVITY
By S. F. SNIESZKO AND G. L. BULLOCK
FISHERY BULLETIN 125
From Fishery Bulletin of the Fish and Wildlife Service
UNITED STATES GOVERNMENT PRINTING O"FFICE WASHINGTON : 1957
For sale by the Superintendent of Documents. U. S. Government Printing Office, Washington 25, D. C. Price 15 cents
Treatment of fm'unculosis with snlfollamides wns unsuccPssful wllell the outbreak was cHused IW sulfonamide-resil;:tllnt nlriantl;: (If the llllthogen. The pat.hogen wns sensUi"e to chloramllhenicol and trentment with this antibiotic resulted in greatl~" reduced nwrtalities. Losses were also gl'el1tI~· I1ffel"ted b~' the strl1in-8pedt1c rel;:il;:tallce or suseeptibilit~· of thl' experimental brook trout to furunculosis.
Sensiti"ity 01' resil;:tl1n(~e (If .-lcrollllll/('s sflllIIOllidrlll to sulfonamides Hnll Ilntibioties was determined in \"itro. ul;:ing "l1riOUl;: liqUid and solhl bl1ctel'iologi- ('al media. A l;:imple method for the df'terminntion in "ill'o of 8ulfl.nl1111ifle- and llntibiotic-l;:ensiti\'ity of .-I., SlIllIIOllidrl" is described for field Ul;:e. A revil;:ed method of treatment of furunculosis is nll;:(1 outlined.
Introduct.ion___ _________________________________________________ 555 Materials and methods_ ________________________ ___________________ 5.55 Results________________________________________________________ 556
Treatment. with sulfonamides and chloramphenicoL . 556 Sensitivity tests with antibiotics____ ___________________________ 558 Sensitivity t,ests wit.h sulfollamides ____________________________ 558
Discussion . _____________________________ 562 Recommendations _______________________________________________ 563 Summary . _________________ ____________ 563 Literature cit,ed . _______________________________________ 564
TREATMENT OF SULFONAMIDE-RESISTANT FURUNCULOSIS IN TROUT AND DETERMINATION OF DRUG SENSITIVITY
By S. F. SNIESZKO, Bacteriologist, and G. L. BULLOCK, Fishery Aid
During the past 2 years, the :Microbiological Laboratory, at Leetown, W. Va.,1 has reeeived an increasing number of reports of outbreaks of furunculosis that failed to respond to treatment with sulfonamides. The problems result.ing from furunculosis, a widespread, bacterial disease of salmonids, and control of the disease have been reviewed in detail by McCraw (1952) and Snieszko (1954a). The latter investigator recommended the use of antibiotics for diseased fish that failed to respond to treatment with sulfonamides. In some of the reeent incidences of furunculosis at the Lee- town station the disease did not respond to sulfon- amide therapy, but control was effected with chloramphenicol. FailUl'e of sulfonamides to con- trol furunculosis suggested that t,he causal organ- ism, Ael'OmOna8 .~almonicida., was resistant, to the drugs used.
Several methods are widely used in the routine determination in vitro of the sensit,ivity of bae- teria to sulfonamides and antibiotics. The result.s obtained with antibiotics are generally accepted as reliable aids in the select,ion of the most prom- ising treatment. Such, however, is not the case with sulfonamides, and "The relation of 1:nl,itI'O sensitivity test to clinical effectiveness is still con- troversial" (Burdette, Plank, and Clapper, 19.55).
The experiment.s presented in t.his paper followed three lines of endeavor: (1) A comparison of the effectiveness of sulfonamide and antibiotic therapy in strains of trout which were either susceptible or resistant to furunculosis. (2) Comparison of the therapy of furunculosis with sulfonamides and chloramphenicol in trout which were suffering from furunculosis caused by sulfonamide-resistant, but chloramphenicol-sensitive, strains of A. sal- monicida. (3) The development of a standard met,hod for laboratory and field use of determin- ing in vitro the sensitivity of A. salmonicida to sulfonamides and antibiotics.
I Post Office. Kearneysville, W. V:l. Note-Approvl'
Many faet-OJ's are capable of affecting in-vitro test,s of microbial sensitivity to sulfonamides; therefore, correct interpretation and application of the result.s of sueh tests will be reliable if a proved method of obtaining reproducible results is used.
As tile result of t.he findings present.ed in this st.udy and praetical expel"ience gained in the treat.ment of t.his disease in trout hateheries over the entire country, a revised pl'oeedure has been evolved and described for the t,reatment of this disease.
The authors wish to express their thanks to Dr. K. E. Wolf for his assistance dming the prepara- tion of the manuscript and for furnishing the strains of A. salmonicida isolated in Iowa, Ut,ah, Minnesotn, and Wisconsin; and to Dr. R. E. Lennon fOJ' supplying the fingerling brook trout from Erwin, Tenn.
MA.TERIALS AND METHODS Treatment with sulfonamides and chloramphenicol
Experiments on the therapy of furunculosis were earried out with two strains of fingerling brook trout (Sa.ll'elimts fontinalis) and a strain of brown t,rout (Salmo tnt.tta). A st,rain of brook trout from Bellefonte, Pa., was selected because it was known to be resistant t.o fUl'Uneulosis (Wolf 1954; Snieszko 1954b), and a strain from Erwin, Tenn., was used because it was suspected to be suscep- tible. The brown trout were from Cortland, N. Y. In genel"al, brown trout are known to be more resistant, to furuneulosis than are most strains of brook trout (McCraw 1952; Wales and Berrian, 1937).
Equal weights of the trout were distributed among stainless steel troughs in which the water was maintained at tempe.ratmes of 12° to 13° C. (54°-55° F.). Infection of the fish was initiated by adding fresh cultures of Ael'omonas salmonicida to the diet, and tl'eatment was started when the first mortality due to furunculosis occurred.
556 FISHERY BULLETIN OF THE FISH AND WILOLIFE SERVICE
Sulfonamides were administered at the rate of 200 milligrams per kilogram of trout per day, or 9 grams per 100 pounds of fish. Chloramphenicol ~ was given at the rate of 75 milligrams of pure antibiot,ic act.ivity per kilogram of fish pel' day, or 3.4 grams per 100 pounds of fish. Treatment, was continued to thc end of the observations recorded in tables 1 to 3. Drugs were mixed with the quantities of food calculat.ed from t.he Cortland Hatchery feeding charts (Deuel et. a!., 1952). A daily reeord of mortality was kept, and most of the dead trout were examined bact.eriologi- cally.
Drug sensitivity tests in vitro
The drug sensitivity tests were performed wit,h 84 cultures isolated from the experiment on t.herapy here described or procured during the past years from other experiment.s or receive.d from other trout. hat.cheries (table 4). The rou- t.ine qualitative sulfonamide and antibiotic sensi- tivity tests were run with commercial multiple sensitivity paper disks 3 on solid bacteriological media. Sensit,ivity to sulfonamides has also been determined in a quantit.a.t,ive manner by prepara- t.ion of serial dilutions of t,hese drugs in liquid bacteriologiea! media.
The following solid bacteriologieal media were used with the paper disks:
1. Leetown standard furunculosis medium having the following composition:
Tryptic digest, of caseill 10 grams. Yeast, extract- ._ ______ 5 grams. Sodium chloride_ _______________ 2.5 grams. Agar .. . 15.0 grams. Wat.er . 1 liter. pH 6.8-i.D.
IA. The same, but. buffered with 0.5 percent of secondary sodium phosphate and adjusted to pH 8.0.
2. Sensitivit.y Test. medium C (Case Labora- tories) .
~A. The same, but. buffered and adjust.ed as No.2.
3. :Mueller Hint.on medium (Bacto). 4. Trypticase Soy agnr (Baltimore Biological
2 Racemic ch)oromy(·,·tin. P:.rk~. DU"is & Co.. was used at a .Iouhl,· rate. hp('au~e thp manufacture,' addSI'd I.hat. it. ha~ ahOll t..~o p,.',·c,-1It the th~l'a peutic "alue of t1w d-rotato,'y i~om,-,'
2 Multidisk~, Case Laboratories. Chicago. Ill.
For the inoculation of the solid media, 24-hour- old cultures in nutrient broth were used. These were streaked wit,h cotton swabs on IO-cm.- diameter petri plates, each containing 20 ml. of medium, and the disks were placed in position.
The quant,it,ative sensitivity test.s were run in liquid media. In oreIer to select the most satis- factory subsLrat.e, t,he following media were tested:
Leet.Q\vn standard furunculosis medium with- out agar.
Vitamin-free easamino acids (Difeo), I per- eent..
Tryptic digest of easein, 1 percent. Nutrient brot.h (Difco). Nut,rient. brot,h (Difco) buffered with 0.5
percent. of secondary sodium phosphat.e and adjusted to desired pH.
Prot.eose pept.one No. 3 (Difco), I percen t.. N-Z-Case (Sheffield Chemical Co.), 1 percent..
Nut.rient. brot.h buffered wit,h sodium phosphate was selected as most promising for our purposes.
Beeause most of t,he sulfonamides are only slight.!)' soluble in wat,er, st.ock solut.ions of sodium salts were prepared by dissolving a known quan- tit.y of sulfonamide in diluted sodium hydroxide. Excess alkali over t.hat, needed for keeping sulfo- namides in solution was neut.ralized with hydro- chloric. ac.id. Stock solutions were sterilized by filt.rat.ion, st.ored under refrigeration, and used within a week. Sel'ial dilutions of 1: 25 01' I: 50 stock solut.ions were c.arried out in 5 ml. of media. The first. tube cont.ained double